PROJECT SUMMARY Chronic wounds affect over 6.5 million patients in the United States alone and result in health costs of more than $25 billion annually. Proper wound healing is the result of a large number of interrelated biological events, which are orchestrated temporally in response to the injury microenvironment. Immediately following injury, a clot is produced which involves the formation of a platelet plug embedded within a fibrin mesh. Platelets bind multiple fibrin fibers and overtime, platelets contract this fibrin mesh through actin driven mechanisms, which contributes to subsequent wound healing by stabilizing the fibrin network, further preventing blood loss, and restoring blood flow past the otherwise obstructive thrombi. Platelet-mediated clot retraction, significantly decreases clot size, alters clot organization, and increases clot stiffness. Increased matrix stiffness has been implicated in activation of important mechanically sensitive pathways involved in wound healing, including Rho GTPase signaling, actin cytoskeleton engagement and mechanical activation of transforming growth factor beta (TGF?), which in turn promote fibroblast migration into the wound bed and extracellular matrix (ECM) production. Importantly, chronic non-healing wounds are characterized by significantly decreased activation of these cellular events. The long-term objective of this proposal is to utilize recently develop platelet-like- particles (PLPs) that mimic this clot retraction feature of native platelets to promote healing in chronic non- healing wounds. PLP-mediated clot retraction occurs via a collective Brownian wrench mechanism to collapse the local fibrin matrix, inducing both global and cell-scale deformations, which ultimately leads to global clot collapse. Particle deformability and high fibrin affinity are critical to achieving PLP-mediated clot retraction. However, the dynamics of PLP-mediated clot retraction are much slower (days) than that of natural platelets (hours). To obtain the benefits of clot retraction in wound repair, it is critical to increase the rate of PLP-mediated clot retraction, to more closely recapitulate the time scale of natural platelets. Therefore, the overarching objective of this proposal is to utilize ultrasound (US) stimulation of PLPs to increase the deformation of PLPs within the fibrin network in order to increase the rate of clot retraction in a finely controlled manner. Our central hypothesis is that 1) US stimulation will increase PLP deformations within the fibrin network, thereby increasing interactions with the fibrin network and the rate and degree of PLP-mediated clot retraction; and 2) enhanced clot retraction will increase clot stiffness, promote fibroblast migration into the wound bed through activation of Rho GTPase signaling, increase ECM production, and increase wound closure rates in vitro and in vivo. We will explore this hypothesis in the following aims: 1) Determine the optimal US sequence to maximize PLP deformation and increase kinetics of PLP-mediated clot retraction. 2) Characterize the effect of PLP-US therapy on wound healing outcomes in vitro and in vivo. The significance of our proposed work is the development of a simple and translatable technology enabling the effective treatment of non-healing wounds.